An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast.
We have developed an activator/repressor expression system for budding yeast in which tetracyclines control in opposite ways the ability of tetR-based activator and repressor molecules to bind tetO promoters. This combination allows tight expression of tetO- driven genes, both in a direct (tetracycline-repressible) and reverse (tetracycline-inducible) dual system. Ssn6 and Tup1, that are components of a general repressor complex in yeast, have been tested for their repressing properties in the dual system, using lacZ and CLN2 as reporter genes. Ssn6 gives better results and allows complete switching-off of the regulated genes, although increasing the levels of the Tup1-based repressor by expressing it from a stronger promoter improves repressing efficiency of the latter. Effector-mediated shifts between expression and non-expression conditions are rapid. The dual system here described may be useful for the functional analysis of essential genes whose conditional expression can be tightly controlled by tetracyclines.
Bellí
G
G
Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, Rovira Roure 44, 25198 Lleida, Spain.
Garí
E
E
Piedrafita
L
L
Aldea
M
M
Herrero
E
E
D001483:Base Sequence; D016213:Cyclins / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D005800:Genes, Fungal; D005809:Genes, Regulator; D017930:Genes, Reporter; D007700:Kinetics; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013752:Tetracycline / Q000494:pharmacology*; D013753:Tetracycline Resistance / Q000235:genetics; D014170:Transformation, Genetic
C082721:CLN2 protein, S cerevisiae; D016213:Cyclins; D004271:DNA, Fungal; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline